Efficient PCR amplification is particularly required in biological applications in which highly sensitive and accurate detection is required. In most primer and probe design programs it is recommended that probes should be designed first and then primers should be designed close to the probes without overlapping sequences (e.g. Primer Express Software Version 2.0, Applied Biosystems, Foster City, Calif.). PCR amplification methods are widely used in the diagnostic industry. Unexpectedly, we discovered that overlapping primer and probe still give efficient PCR, expanding probe and primer design opportunities particularly in challenging sequence environments. The PCR method and its clinical applications have been disclosed (U.S. Pat. No. 4,683,202; Lynch J R, Brown J M. J Med. Genet., 27:2-7 (1990); Yang S, Rothman R E. Lancet Infect Dis., 4:337-48 (2004))
The schematic representation of the overlapping probe and primer in a novel amplification method of the invention is shown in FIG. 1. FIG. 1a) and b) show a schematic representation of primer and probe location on the target, with a primer sequence overlapping with a probe sequence. FIG. 1c) shows a schematic representation of two overlapping primer sequences. FIG. 1d) shows a schematic representation of a probe sequence overlapping with both primers.
5′-Minor groove binder (MB)-Quencher (O)-oligonucleotide-Fluorophore (Fl)-3′ or 5′-MB-Fl-oligonucleotide-Q-3′ or 5′-Fl-oligonucleotide-Q-MB-3′ probes have not been overlapped with primers. It is suggested in the PCR amplification literature that “one should avoid complementarity at 3′-ends of primer pairs as this promotes the formation of primer-dimer artifacts and reduces the yield of the desired products (Innis, M. and Gelfand, D. Optimization of PCR in Innis, M., Gelfand, D., Sninsky, J. and White, T., Editors. PCR PROTOCOLS A GUIDE TO METHODS AND APPLICATIONS. Academic Press, San Diego, Calif. pages 3-12, 1989.) Since the probe and primer have overlapping complementary sequences and available 3′ sequences, it was expected the 5′-MB-Q-oligonucleotide-Fl-3′ or 5′-MB-Fl-oligonucleotide-Q-3′ or 5′-Fl-oligonucleotide-Q-MB-3′ probes overlapping with a primer would give poor amplification. However, unexpectedly it was observed that efficient amplification occurs in the case of FIG. 1b. Without being bound to a theory, we suspect that the probe containing a MB ligand, Q and a Fl has a tight conformation in solution where these components are in close proximity. We have shown that such a probe in solution is quenched from temperatures ranging from 25 to 95° C., suggesting that overlaps from 1 to about 7 bases are not enough to overcome the in solution stable conformation of the probe to allow hydrogen bond formation. In FIG. 1a the Primer-Probe dimer should not give rise to any artifacts. The 3′-end is not available for priming. Surprisingly, this type of design can provide good amplification.